The binding of dansylated initiation factor 3 to the 30-S subunit of Escherichia coli: a fluorimetric and biochemical study.
Initiation factor 3 (IF-3) has been labelled using dansyl (1-dimethylaminonaphthalene-5-sulphonyl) chloride under conditions designed to preserve the biological activity of the factor. The sites of modification of the IF-3 have been determined by peptide mapping and sequencing: about six lysines (73, 80, 91, 96, 99, 112) react in various proportions. However, if an IF-3 molecule bears more than one dansyl group on average then its activity is lost. The extent of incorporation is proportional to the amount of dansyl chloride used in the reaction. Spectrofluorimetric analysis of the dansyl-IF-3 leads to the following conclusions. (a) The motion of the dansyl label does not change greatly upon binding to the 30-S subunit. (b) The label is not close enough to any tryptophan group of the ribosome in the 30-S-subunit . IF-3 complex to allow energy transfer. (c) The IF-3 chain is folded so as to bring the tyrosine groups close to the dansyl-binding sites. (d) The stoichiometry of the binding of IF-3 to 30-S ribosomal subunits is close to 1:1 and the binding constant is 2 x 10(7) M-1. IF-3 also binds non-covalently the fluorescent indicator 8-anilinonaphthalene 1-sulphonate (ANS) with an apparent binding constant of approximately 8000 M-1. An interaction between ANS and poly(A-U-G), both bound to IF-3, was demonstrated. Combining these results with those for dansyl-IF-3 leads to a model for the interaction between IF-3 and the 30-S subunit involving a combination of 'hydrophobic' and electrostatic attraction between the factor and ribosomal RNA.
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D007700:Kinetics; D010446:Peptide Fragments / Q000032:analysis; D010448:Peptide Initiation Factors / Q000378:metabolism*; D011485:Protein Binding; D012269:Ribosomal Proteins / Q000378:metabolism*; D013861:Thiocyanates; D014364:Tryptophan / Q000032:analysis; D014443:Tyrosine / Q000032:analysis
D005452:Fluoresceins; D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D012269:Ribosomal Proteins; D013861:Thiocyanates; C030936:dansylated initiation factor 3; D014443:Tyrosine; D014364:Tryptophan; D016650:Fluorescein-5-isothiocyanate